1. Field of the Invention
The present invention relates generally to microarray spotting instruments and, more particularly, to a method and apparatus for detecting the presence of pins at particular locations in a printhead of such instruments.
2. Description of Related Art
As is well known (and described, e.g., in U.S. Pat. No. 5,807,522 issued to Brown et al. and in “DNA Microarrays: A Practical Approach,” Schena, Mark, New York, Oxford University Press, 1999, ISBN 0-19-963776-8), microarrays are arrays of very small samples of purified DNA or protein target material arranged as a grid of hundreds or thousands of small spots on a solid substrate. When the microarray is exposed to selected probe material, the probe material selectively binds to the target spots only where complementary bonding sites occur, through a process called hybridization. Subsequent quantitative scanning in a fluorescent microarray scanner may be used to produce a pixel map of fluorescent intensities (See, e.g., U.S. Pat. No. 5,895,915 issued to DeWeerd et al.). This fluorescent intensity map can then be analyzed by special purpose quantitation algorithms, which reveal the relative concentrations of the fluorescent probes and hence the level of gene expression, protein concentration, etc., present in the cells from which the probe samples were extracted.
The microarray substrate is generally made of glass that has been treated chemically to provide for molecular attachment of the spot samples of microarray target material. The microarray substrate is generally of the same size and shape as a standard microscope slide, about 25 mm×75 mm×1 mm thick. The array area can extend to within about 1.5 mm of the edges of the substrate, or can be smaller. The spots of target material (typically DNA) are approximately round. The spot diameter usually varies from about 75 microns to about 500 microns, depending on the dispensing or spotting technique used. The center-to-center spacing between the spots usually falls into the range of 1.5 to 2.5 spot diameters.
FIG. 1A, which is not drawn to scale, is a top view of a prior art microarray 100. In this drawing, each of the circles represents a tiny spot of target material that has been deposited onto a rectangular glass substrate 101. The spots are shown magnified relative to the substrate 101. For convenience of illustration, only a few spots (a six by six array) are shown covering a small area of the substrate. However, thousands of spots are usually deposited in a typical microarray, and the spots may cover nearly the entire substrate.
There are several well known methods of depositing the spots onto the substrate of a microarray, and instruments that deposit the spots are typically referred to as “spotting instruments.” One such instrument works similarly to an ink-jet printer, where a flew microliters of sample are aspirated by vacuum out of a sample reservoir into a hollow tube or needle. One or more droplets of the sample are then ejected from a nozzle end of the tube onto the substrate to form a spot.
Other spotting instruments use pins as spot dispensers. This method of spotting generally comprises (1) dipping a pin into the liquid sample in a sample reservoir where some amount is taken up by capillary action or surface tension, (2) moving the pin to a predetermined location above a microarray substrate (typically using a robotic arm), and (3) lowering the pin until its tip makes contact with the substrate. Some of the sample material is transferred to the substrate by either inertia or surface tension to form a microarray spot.
Pins are commercially available in several distinct forms. The simplest pins are solid. FIG. 1B shows an example of such a prior art solid pin 102, which includes a pinhead 104 and a shaft 106. Both the pinhead 104 and the shaft 106 are generally cylindrical and coaxially arranged. The diameter of the shaft 106, which is about 1 mm, is less than the diameter of the pinhead 104. One end or tip 107 of the shaft 106 is tapered or sharpened, and the other end is attached to or integral with the pinhead 104. The tip typically includes a small flat, and the area of the flat (along with the surface properties of the sample liquid and the microarray substrate) determine the size of the spot that the pin will form. Solid pins are simple and robust, but in being dipped into the target material in a well typically only take up enough material to form one spot. This then requires the spotting instrument to dip the pin once for every microarray spot that is to be printed.
Other types of known pins can hold enough target material from a sample reservoir to form several, perhaps even hundreds, of spots before they need to be re-dipped in the reservoir. One such type of pin (not shown) is formed from a hollow cylindrical tube with an axial slot cut in the tip ( See, e.g., U.S. Pat. No. 5,770,151 issued to Roach et al.). This pin draws up sample liquid into the tube and slot by capillary action, and deposits it in small amounts onto the substrate by capillary action upon contact with the microarray substrate. The uptake volume of the pin is sufficient to form dozens of spots by subsequent contact with other microarray substrates in the batch being processed.
Another type of multi-spot dispensing pin is a solid “two-piece” pin (not shown), which has a gap or slot at the shaft tip. This type of pin draws fluid into the gap or slot by capillary action, and deposits a small amount onto the substrate by the inertia of the fluid when the pin is rapidly decelerated by lightly tapping it on the substrate. Again, the amount dispensed to form a spot is small compared to the sample uptake volume, so that each dip of the pin into sample liquid takes up enough sample material to form dozens of spots.
Yet another type of multi-spot dispensing pin available, e.g., from TeleChem International, Inc., is similar in appearance to the two-piece pin, but operates somewhat differently. These pins are solid, with a pyramidal taper at the tip that ends in a small square flat. A small slot is cut across the tip, providing a reservoir for holding sample liquid. The tip of the pin is then squeezed or bent slightly to bring the two segments of the slotted tip closer together. In use, these slotted pins are dipped into sample liquid, where a small quantity (e.g., a fraction of a microliter) of sample is taken up into the slot by capillary action. The specific geometry and material of the pin also causes a very small amount of liquid in the slot to wick out onto the two segments of the split pin tip. Then, the pin is brought into contact with the microarray substrate where capillary action attracts the portion of sample that is on the pin tip and forms a spot.
Each type of pin must be manufactured to very precise tolerances to insure that each spot formed by the pin will be of controlled size. As a result of these demanding specifications, the pins are expensive (e.g., a single pin typically costs several hundred dollars). Also, the pins are very fragile given that the pin tips are so small and precisely shaped. Consequently, to avoid damage, the tips can only be subjected to a very small force when they are placed in contact with the substrate or any other solid object.
Spotting instruments typically form microarrays in batches. For example, in a single “run”, a spotting instrument may form up to 100 identical microarrays. After forming enough spots to complete the batch of microarrays being spotted, the pins generally need to be washed (to remove any excess liquid target material), and then dried before they can be dipped into another reservoir of target material. So the process of forming microarrays with a “pin-type” spotting instrument includes steps of (1) positioning a pin over a reservoir of target material; (2) dipping the sharp end of the pin into the reservoir; (3) withdrawing the sharp end of the pin from the reservoir; (4) moving the pin over a selected location within the active area of a microarray; (5) lowering the pin to bring the sharp end of the pin into contact with the microarray substrate to form a single spot of controlled size at the selected location; (6) repeating steps (4) and (5) until the pin's supply of target material is exhausted or until the desired number of spots have been placed on the batch of microarrays being produced; (7) raising the pin to separate the sharp end of the pin from the substrate; (8) washing the pin by either placing the pin in a stream of cleaning solution or by dipping the pin into a reservoir of cleaning solution; and (9) drying the pin. The spotting instrument repeats all of these steps numerous times to form a single microarray.
Since microarrays typically include thousands of spots, using only a single pin to form the microarray would be extremely time consuming. Accordingly, spotting instruments are typically designed to simultaneously manipulate several pins. FIGS. 1C, 1D, and 1E show side, top, and perspective views, respectively, of a simplified printhead 110 that can simultaneously hold up to sixteen pins 102. Printhead 110 is a block of material, typically metal, that includes an array of sixteen through-holes or apertures 112. The apertures 112 are slightly larger than the outer diameter of the pin shafts 106 so the shafts can extend through the apertures 112. The apertures 112 are also smaller than the outer diameter of the pinheads 104 so that when the pin shaft is dropped into one of the apertures 112, the pinhead 104 will be supported by the upper surface of the printhead 110. The pins are thereby “slip-fit” into the apertures of the printhead. FIGS. 1F and 1G show side and top views, respectively, of the printhead with sixteen pins mounted therein.
FIG. 1H illustrates printhead 110 being lowered to place the tips of the pins 102 into contact with substrate 101 and thereby simultaneously forming sixteen spots of target material on the substrate. As shown, the printhead is generally lowered about 1 mm further than needed to place the tips of the pins in contact with the substrate 101. The slip-fit allows the upper surface of the printhead to be lowered beneath the bottom of the pin heads without imparting any significant force to the tips of the pins. The printhead is preferably lowered sufficiently slowly so that the force applied to the tips of the pins (1) is principally determined by the weight of the pin plus a minor additional force due to the friction of the slip-fit and (2) is not significantly affected by inertial forces. The act of lowering the printhead to place the tips of the pins in contact with the substrate and thereby forming spots on the microarray is commonly referred to as “printing.”
Pins sometimes get stuck in an ‘up’ or raised position in the printhead, ie., the position shown in FIG. 1H. Pins generally weigh 0.4-1.0 grams and rely on gravity to pull them down so that the pin head rests on the top surface of the printhead when the printhead is lifted (as shown in FIG. 1F). Friction resulting from, e.g., dirt or finger oil on the pin shaft or a slightly bent shaft, can prevent the pin from properly falling down to its rest position. If a pin gets stuck in the up position, it is not useful for printing. A need thus exists for a method and apparatus for quickly and accurately determining whether there are any pins in the printhead that are stuck in an up position.
Commercially available printheads provide between 4 and 72 apertures, thereby accommodating between 4 and 72 pins. Commercially available reservoirs provide a plurality of wells, or individual reservoirs, and permit each pin mounted in a printhead to be dipped into a separate well. Two popular reservoirs useful for producing microarrays are the “96-well plate” and the “384-well plate.” Each of these plates provides a rectangular array of wells, each well being capable of holding a unique sample of liquid target material. FIG. 1I shows a top view of a 96-well plate. In 96-well plates, the centers of the individual reservoirs are separated by 9.0 mm, and in 384-well plates, the centers of the individual reservoirs are separated by 4.5 mm. The centers of adjacent apertures in commercially available printheads are correspondingly separated by either 9.0 or 4.5 mm. Pin-type spotting instruments generally include mechanisms for holding or manipulating one or more plates (e.g., either 96-well or 384-well), a printhead, a robotic manipulator for controlling the movement of the printhead, mechanisms for holding a plurality of substrates, a pin washer, and a dryer.
In many spotting instruments, the printhead is readily accessible to the user, and the user configures the printhead with the number and arrangement of pins as desired. Often, a printhead that can accommodate 32 or more pins is populated with only 4, 8, or 16 pins. The printhead may not be fully populated for several reasons. First, the user might desire a compact pattern of spots in the finished microarray. For instance, an array made with a fully populated 48-pin printhead would probably be 18 mm×54 mm in extent. That size of an array is large enough to require a large amount of fluorescent probe material to cover it, and to require special care to ensure that the hybridization reaction of the probe is uniform. Second, the pin spacing in the printhead might not correspond to the well spacing in the well plates. For example, 96-well plates have wells on 9.0 mm centers, and 384-well plates have wells on 4.5 mm centers. If a printhead with 4.5 mm pin spacing is used with a 96-well plate for spotting, then only every fourth hole in the printhead can be populated with a pin, or else all the pins will not be aligned with wells in the plate. Third, the user might not have enough pins available to fill a printhead. Pins can easily be damaged, and are expensive. Many users do not invest in a complete set of pins while they arc initially qualifying their microarray process, and/or may not immediately replace a damaged pin.
Spotting instruments include robotic manipulator arms that are driven through a series of repetitive motions by one or more computer controllers. The printhead and/or microarray sample plates and/or microarray substrates are moved by robotic arms relative to one another in three dimensions (i.e., X, Y and Z axes). As previously mentioned, a spotting cycle includes sample uptake (dipping pins in particular wells of a particular plate), spotting (depositing spots of the sample in particular locations on one or more microarray substrates), then washing and drying the pins on the printhead. Each subsequent printing cycle is performed with the printhead's pin positions indexed to dip into the next series of wells on the plate (or on the next plate) and to print on the next spot positions on a substrate. The instrument's controller keeps track of and controls the indexing of positions of the sample uptake and printing motions for each cycle.
In commercially available pin-type spotter instruments, for a controller to perform these positioning tasks, the parameters of which printhead positions are occupied by pins must be known and entered into the controlling program. In known spotting instruments, this information is manually entered by the user, either as alphanumerical information or using a graphical user interface display. If the user mistakenly enters incorrect pin location information, spotting errors and even damage to the pins can occur. Pin location can usually be readily determined by visual inspection when the printhead is small and with few pins. However, with a printhead having a capacity of 32 pins or greater and dozens of pins, it is tedious and error prone to determine pin positions and enter them manually into the controlling computer. A need thus exists for a method and apparatus for quickly and accurately determining pin positions in pin-type spotter instruments.